EMPIRI INC.
Frequently Asked Questions
For general FAQs regarding how we do things, click here
For FAQs on single cell sequencing procedures, click here
General FAQs
Q: How do I request a quote?
A: To request our services, click here
Q: How do I submit the EMPIRI sample form (to provide detailed information about my samples)?
A: This information is included in your quote.
Q: How can I monitor the progress of my project?
A: To request the status of your project, click here to email us with your order number or project ID
Single Cell Sequencing FAQs
Q: What is single-cell RNA-Seq?
A: Single-cell RNA-Seq provides transcriptomic information from thousands of cells from each sample. This level of throughput analysis enables researchers to understand how many different cell types are present in a sample, what genes and pathway activation states distinguish different cell types, and how different experimental or clinical manipulations change different cell types in numbers and molecular phenotypes.
The workflow for Single-Cell RNA-Seq is outlined on our services webpage. Fresh tissue/cell samples are delivered in transport media to EMPIRI. At EMPIRI, single cell suspension is generated, and if necessary, dead cells are removed prior to single-cell capture (see below for more information). Single cells are then captured by the 10x Genomics Chromium™ Controller in individual droplets. 5' transcriptome sequencing libraries are then generated and sequenced (standard analysis targeting 30,000 - 50,000 reads per cell). Please visit 10X Genomics website for more detailed information.
Q: What is the difference between bulk RNAseq and single-cell RNA-seq?
A: Standard or bulk RNA-Seq measures averaged genes expression from all cell types present in the sample. Bulk RNA sequencing usually samples millions of cells and detects 15,000-18,000 transcripts in a typical experiment. However, it is impossible to definitively assign from which cell each transcript originated. In contrast, in single cell RNA-seq, individual cells are barcoded during the capture step, allowing definitive assignment of each transcript to specific cells. On the other hand, high throughput single cell sequencing using the 10x Genomics 5' transcriptome reagents detects ~2000 - 4000 transcripts per cell and only ~91bp of the transcript from the 5' end.
Q: Can you sequence samples that are shipped overnight?
A: Yes but be aware that gene expression patterns change during transport and storage, and certain fragile cell types, such as neutrophils, will be lost as they can die during long-term storage. In general, we try to dissociate and capture cells as quickly as possible, usually <6 hours after delivery to EMPIRI.
Q: How many cells can be sequenced from each sample?
A: Depending on the experimental goal and sample availability, the total number of cells captured and analyzed can vary from 500 to 10,000 cells per sample. Higher cell numbers will result in higher doublet rates (two or more cells per droplet) and lower sequencing saturation rates. Therefore, it is not recommended. If the analysis of higher numbers of cells is desired, more cells can be captured in additional lanes (and cost). We typically recommend targeting 3,000 - 6000 cells per sample for most experiments.
Q: How many reads do I need for my experiment?
A: The number of reads required depends upon the cell type and experimental goal. Generally, we recommend 50,000 reads per cell.
Q: How do dead cells impact data quality?
A: Presence of dead cells and cellular debris can impact single-cell RNA-sequencing in many ways: compromised droplet capture, increased ambient RNA, missed sequencing targets, and suboptimal results. We aim for >85% viability. Samples with less than 70% viability may undergo dead cell removal (DCR) unless the starting cell number is very low and the client is willing to move forward without DCR. DCR can result in significant cell loss depending on the method used and prior treatments of the sample.
Q: What types of data analysis are available?
A: We start with the Cell Ranger software from 10x Genomics for the basic alignment and analysis. We also perform custom analysis based on the Seurat method that is included in our service fee, which includes the following:
read alignment, feature-barcode matrix, digital gene expression matrix, clustering, differentially expressed gene list, t-SNE and UMAP projections (shown below), Cloupe file for further exploration by the investigator. Loupe Cell Browser is a free download from 10x Genomics to explore your data, including interactive tools for finding significant genes, identifying cell types, and exploring substructure. More information, tutorials, and demos can be found at 10X Genomics.
Q: How is data delivered?
A: EMPIRI will recommend the best data delivery option based on project details. Data delivery options include Secure file transfer protocol (SFTP), customer cloud account, physical external drive (additional charges may apply).
Q: How do I contact EMPIRI for a technical consultation about my project?
A: EMPIRI's Single Cell Team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation. Contact us for additional information.
Q: How do I request a quote / What is the price for Single-Cell RNA-Seq?
A: Pricing varies based on the details of the project. Please submit a request here.
Q: How should I prepare and send my samples?
A: Sample Type: Fresh tissue or single cells in transport media on ice. Recommended Amount: approximately 10^5 - 10^6 viable cells. Minimum Amount: 20,000 cells. Buffer: Transport media or cell culture media used. Tubes/Plates: 1.5m, 2ml, 15ml, or 50ml tubes. Shipment Method: EMPIRI will contact you to coordinate sample pick up or drop off.
Q: What happens if my samples do not meet your starting material requirements?
A: We will contact you if the total viable cell number is <5000 before loading the chip.
Q: How do I submit the EMPIRI sample form (to provide detailed information about my samples)?
A: You may provide additional details along with your order submission request. To provide additional info at a later stage, please email our customer service department with your order or project ID.
Q: How can I monitor the progress of my project?
A: Projects with standard analysis usually take about 6-8 weeks. An expedited turnaround is possible at additional cost. A more accurate timeline will be provided upon sample submission. To request an update during processing, please email customer service with your order or project ID.
Q: How long do you hold samples?
A: We hold sequencing data for 1 month after project completion and data transfer. Longer storage in the EMPIRI cloud space requires additional charge. It is the responsibility of the client to download or make arrangements for all data for publication and long term storage.
Q: Number of biological replicates for single-cell RNA sequencing?
A: It depends on the biological question and variability of samples within a study.
Q: Can Single Cell Sequencing be performed on any species?
A: In theory, it is possible to analyze any species with the reference genome. However, we currently offer only mouse and human sample analysis.
Q: Any best resource (Online- paid/free) for learning Single-Cell RNA-Seq Data Analysis online?
A: Here are a couple of resources that can help. However, we do not currently endorse any as a company. You must find the best available resource that best suits your needs. https://github.com/hemberg-lab/scRNA.seq.course ; https://github.com/seandavi/awesome-single-cell
Q: How is the accuracy of single-cell sequencing?
A: Nature magazine had its cover devoted to 'Single-Cell Biology', in the 6 July 2017 issue, Vol 547, Nº 7661. Click here >
E-Slices FAQs
Q: What is an E-Slice ?
A: E-slice is EMPIRI's proprietary, 3D tumor slice culture method. It retains intact tissue microenvironment and is cultured in a serum-free, chemically defined medium to minimize altering the native tumor tissue microenvironment and cell states. It has been validated to accurately predict patient responses in clinical trial settings and can be used with any solid tumor types from human or mouse origin.
Q: Can you perform tumor slice cultures from biopsy needle cores?
A: Yes, we can as long as they contain viable cells.
Q: How long can you keep your cultures?
A: It depends on the tissue but we have maintained good viability with multiple tumor types for up to 30 days ex vivo.
Q: Does the E-slice culture medium contain serum?
A: No, we use a serum-free defined medium to minimize uncontrolled and artificial signaling in patient tumors.
Q: Can you take longitudinal measurements?
A: Yes, we routinely measure treatment responses at 3-4 different time points from the same tissue. By doing so, we can detect dynamic changes in response to treatment and the emergence of acquired therapy resistance in some samples.
Q: Which tumor types can you culture?
A: Thus far, the E-slice platform has worked with every tumor type we tested. On rare occasions, we observed very slow to no growth from some patient samples but these samples may have contained necrotic or normal tissues or just very slow-growing tumors.
Q: Can E-slice be used to discover biomarkers?
A: Yes, since we use a serum-free, defined medium, it is straightforward to detect secreted proteins or other molecules in the conditioned medium or treated tissues.
Q: Can we test different combinations or sequences of drug treatment using E-slices?
A: Yes, we can test single or multiple agents simultaneously or in different sequences as the study requires.
Q: Can you fix the tissue for histological analysis or other analysis?
A: Yes, we can fix the tissues or make RNA, DNA, or protein lysates from samples at any desired time points.
Q: Can you use E-slices to stratify patients or predict clinical response?
A: Yes, we have validated that our method can accurately predict patient responses to different targeted therapies and immunotherapies in clinical trial settings.
Q: Does the E-slice platform work with mouse tumors or PDX models?
A: Yes. Our method was optimized through over 300 experiments with mouse and human tumors.